Monocytes/macrophages are crucial cell types in inflammatory processes in many diseases including musculoskeletal diseases. Osteoclasts, (cells derived from monocytes/macrophages) have the unique capacity to resorb bone and thus play a central role in the homeostasis of musculoskeletal system. In Inflammatory osteolysis (resorption or dissolution of bone due to inflammation) observed in disorders such as rheumatoid arthritis, osteoclasts erode periarticular bone, leading to joint collapse and disfigurement. Tumor necrosis factor (TNF) is one of the dominant cytokines that play a critical role in the pathophysiology of these inflammatory disease conditions and excessive TNF signaling in macrophages has been attributed to an increased osteoclastic recruitment and activity in diseases such as arthritis. TNF is also one of the most potent pro-inflammatory cytokines that act on monocytes/macrophages. Thus understanding the biochemical mechanisms by which TNF stimulates signaling pathways in monocytes/macrophages and the mechanisms by which TNF-induced signaling affects survival of macrophages is of vital importance for therapeutic targeting in arthritis. We demonstrate in preliminary data that G-protein coupled receptor kinases (GPCR kinases or GRKs) -2 and -5 play novel and crucial roles in the signaling of TNF receptors in macrophages. This suggests that GRKs could potentially play a key role in the pathogenesis of arthritis. GRKs are serine/threonine protein kinases originally discovered for their ability to phosphorylate agonist-occupied G-protein coupled receptors (GPCRs). Role of GRK2 and 5 in TNF signaling represents a novel signaling paradigm and thus understanding the mechanisms by which these GRKs regulate TNF signaling in macrophages and in TNF-mediated diseases such as arthritis deserves further investigation. In preliminary proteomics experiments we have identified death-associated protein kinase (DAPK) as a novel interacting protein of GRK2. DAPK is a critical signaling protein involved in the regulation of TNF-induced growth/apoptosis in many cell types (3). In this grant, we propose to expand our preliminary findings on the novel roles of GPCR kinases in TNF signaling in macrophages. The central hypothesis of this application is that GPCR kinases play a crucial role in TNF signaling and macrophage survival and thus are critically involved in the pathogenesis of arthritis. To test this hypothesis we propose to examine the following specific aims: 1. Determine the role of GRK2 interaction with death-associated protein kinase (DAPK) in TNF-induced macrophage survival/apoptosis. 2. Identify and characterize novel GRK2-interacting signalosomes upon TNF stimulation in macrophages. 3. Determine the role of GRK2 and 5 in a mouse model of arthritis. Biochemical, proteomic and in vivo approaches proposed here will answer critical questions on the role of GRKs in cellular, biochemical and pathophysiological basis of TNFR signaling in macrophages which will form the basis for identifying molecular/therapeutic targets for TNFR signaling in inflammatory disease conditions such as arthritis. RELEVANCE: Tumor necrosis factor (TNF) acts on macrophages and plays an important role in the development of various diseases including arthritis by activating signaling pathways. Major goal of this application is to understand how these signaling pathways are regulated by TNF in macrophages and in arthritis.